System and method of perfusion 3D cell culture

Abstract

A 3D perfusion biochip system and method for more accurate modeling of in vivo environmental conditions and cellular behaviors. The biochip includes a cavity for containing culture media and cells. A plurality of series-coupled capillary tubes cross the cavity. In some embodiments, the capillary tubes are arranged in a swirl pattern. In some embodiments, the capillary tubes include micropores. In some embodiments, the biochip includes multiple parallel layers of series-connected capillary tubes, where the capillary tubes of each layer are independently arranged in one of an outside-in swirl pattern, an inside-out swirl pattern, or an S pattern.

Description

SYSTEM AND METHOD OF PERFUSION 3D CELL CULTURECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This Application claims priority to U.S. Provisional Patent Application No. 63 / 554,371 entitled “System and Method of Perfusion 3D Cell Culture,” filed on February 16, 2024, the entire disclosure of which is incorporated herein by reference.FIELD

[0002] This disclosure relates to cell biology and / or tissue engineering, and more particularly, to systems and methods for mimicking a three-dimensional (3D) biological environment for cells using a perfusion-based 3D biochip for a 3D cell-culture model.BACKGROUND

[0003] Recent drug discovery efforts have focused on incorporating in vitro cell models that better mimic the in vivo conditions found within a target patient. Conventionally, a predetermined quantity or concentration of model cells is seeded onto a coated microplate well. The microplate is incubated to encourage the cells to attach in a two-dimensional (2D) monolayer before performing a prescribed assay. While this may provide some improvements over biochemical and immortalized cell lines, culturing cells in this 2D manner may be problematic, at least because 2D cell-culture models typically do not accurately model in vivo environmental conditions and / or cellular behaviors.

[0004] For example, studies of 2D cell-culture models have shown that attrition rates of drug candidates for cancer can be as high as approximately 95%. Further, in vitro drug efficacy values can fail to translate to clinical environments. Still further, unforeseen toxicity issues can arise. First, it is believed that in 2D cell-culture models, some extracellular matrix (ECM) components are missing, and therefore, certain cell-to-cell and cell-to-matrix interactions do not occur. These components and interactions are critical to cell differentiation, cell proliferation, and / or cellular function that normally occur in vivo. Second, it has been found that the environment of a 2D cell-culture model may not accurately mimic a 3D in vivo environment where cancer cells reside, because the 2D environment does not allow for areas of hypoxia, heterogeneous cellpopulations, varying cell proliferation zones, ECM influences, soluble signal gradients, and / or differential nutrient and metabolic waste transport.

[0005] 3D cell-culture models may be utilized to overcome many limitations of 2D cellculture models because 3D cell-culture models more closely mimic the features and environments of complex in vivo conditions. For example, studies have shown that tumor cells of specific cell lines, evaluated using 3D cell-culture models, are less sensitive to anti-cancer agents than when the same tumor cells are cultured using 2D cell-culture models.

[0006] One way to build 3D cell-culture models is to form 3D scaffolds out of porous polymeric and / or biologic materials. At least one study determined an optimal pore size (e.g., pore diameter) for 3D scaffolds of cell-culture models of approximately 100 - 400 pm. See Han, et. al, “Effect of Pore Size on Cell Behavior Using Melt Electrowritten Scaffolds,” Frontiers in Bioengineering and Biotechnology, Volume 9, Article 629270, July 2021. This result was based on achieving good cell nutrition, cell-growth space, and cell-scaffold interaction (the result depended on the specific cell lines).

[0007] Consistently controlling the size and shape of pores to achieve repeatable results requires a high precision manufacturing method. For example, to consistently create pores with diameters of 100 pm typically requires a manufacturing method having sub-20 pm resolution, such as micro 3D printing. In particular, projection micro stereolithography (PpSL) and two-photon polymerization are micro 3D printing techniques that are capable of consistently creating complex 3D structures. Further, these printing techniques can create 3D structures from biocompatible and / or biodegradable polymers, such as poly-ethylene glycol (PEG) and poly lactic acid (PLA). After polymerization, these polymers may be hard (e.g., rigid) or soft (e.g., flexible).

[0008] Cell cultures can be described in terms of reaction kinetics. In steady state, the process of cells consuming metabolites (e.g., cell nutrients) is often described by the Michaelis- Menten equation:where v is the velocity of the reaction, Vmax is the maximum uptake rate of metabolites, KM is the metabolite concentration when the uptake rate is half of the maximum (the Michaelis constant), and [S] is the concentration of metabolites. In Michaelis-Menten kinetics, the consumption behavior follows first order kinetics at low concentration. This means the consumption rate is proportional to the concentration. As the concentration of the metabolite increases, theconsumption behavior will gradually become zero order kinetics. This means the consumption rate is near or equal to the maximum velocity and is independent of metabolite concentration. This is because the cells eventually become saturated and therefore their intake of metabolites reaches a plateau.

[0009] In an in vivo environment, when the cell concentration is on the order of IO10cells / ml, the cells tend to stay less than 100 pm from blood capillaries. To supply metabolites to such a high-density cell cluster, a high density of nutrient transport passages (capillaries) is required.SUMMARY

[0010] The disclosed embodiments provide for a perfusion-based 3D biochip and a method of using the same. More specifically, the disclosed embodiments provide for a perfusion-based 3D biochip and a method of use that can more accurately model in vivo biological conditions and cellular behaviors for studying cell biology and tissue engineering, for example, high-density cell- to-cell interaction, high-density cell-to-ECM interaction, high-density cell-to-drug response, biomaterial development, and new drug development.

[0011] Embodiments of the disclosed perfusion-based 3D biochip for 3D cell cultures comprise a capillary system to promote 3D cell-to-cell and cell-to-ECM interactions as well as to promote transportation of metabolites and metabolic waste. Some embodiments of the 3D biochip are well suited for high cell-density 3D cell cultures because, for example, capillary tubes of the biochip can deliver nutrients to the cells to enable even densely packed cells to reach the maximum uptake rate of nutrients, thereby promoting healthy growth and / or maintenance of the cells. Some or all of the structures of the disclosed embodiments of the 3D biochip, for example the capillary tubes, may be created via PpSL micro 3D printing and two-photon polymerization techniques.

[0012] Some embodiments of the 3D biochip comprise: a cavity on an upper surface of the biochip; N > 2 capillary tubes fluidically coupled in series and crossing through the cavity, comprising an initial capillary tube fluidically coupled to an inlet of the biochip and a final capillary tube fluidically coupled to an outlet of the biochip; and N-l elbows, wherein each ithelbow connects an ithcapillary tube to an (i+l)thcapillary tube, where 1 < i < N and N is an integer. In some embodiments, the capillary tubes are arranged in an outside-in swirl pattern; an inside-out swirl pattern; or an S pattern. In some embodiments, there are multiple layers of N capillary tubesand N - 1 elbows. In various embodiments, part or all of at least one elbow may be disposed within the cavity, within a body of the biochip (e.g., a bulk material portion of the biochip), or external to both the cavity and the body of the biochip, and further, part or all of at least one elbow may be adapted to exhibit capillary behavior (e.g., capillary-like) or it may be adapted to not exhibit capillary behavior (e.g., non-capillary).BRIEF DESCRIPTION OF THE DRAWINGS

[0013] A more complete appreciation of the present disclosure and many of the attendant advantages thereof will be readily obtained as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings.

[0014] FIG. 1A shows a perspective view of an example of a 3D biochip from above and FIG. IB shows a perspective view of the 3D biochip from below.

[0015] FIG. 2A shows a cross section of the 3D biochip of FIG. 1A taken along line A-A including a representation of a flow of culture therein and FIG. 2B shows a cross section of the 3D biochip of FIG. 1A taken along line B-B including a representation of a flow of culture therein.

[0016] FIG. 3A shows a cross section of the 3D biochip of FIG. 1A taken along line C-C and FIG. 3B shows a cross section of the 3D biochip of FIG. 1A taken along line D-D.

[0017] FIG. 4 shows a cross section of the 3D biochip of FIG. 1A taken along line E-E as part of an example of a system for studying cell biology and / or tissue engineering.

[0018] FIG. 5 is a flowchart of an example of a technique for building a 3D cell-culture model.DETAILED DESCRIPTION

[0019] The present disclosure may be more readily understood by reference to the following detailed description and the accompanying drawings, which form a part of this disclosure. This disclosure is not limited to the specific devices, methods, conditions, or parameters described and / or shown herein, and the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of disclosed embodiments or inventions. For example, “top,” “bottom,” “left,” “right,” “clockwise,” and “counterclockwise” may be used as specific examples of generally opposite orientations or directions, respectively. Also, as used in the specification and including the appended claims, thesingular forms “a,” “an,” and “the” include the plural, and reference to a particular numerical value includes at least that particular value, unless the context clearly dictates otherwise.

[0020] The following numerals are used to describe various features of the embodiments: biochip 10, cavity 20, opening 30, inlet 40, outlet 50, capillary tube(s) 100, support rib(s) 110, elbow(s) 120, channel 130, system 200, basin 210, window 220, microscope 230, input port 240, inlet pump 250, inlet seal 260, suction outlet 270, culture-media bath 280, cells 290.

[0021] FIGS. 1A-1B show an embodiment of an example of a perfusion-based 3D biochip 10 having a bowl-shaped cavity 20 defined on a top surface of the biochip 10, such that a flared top rim of the cavity 20 is adjacent to the top surface of the biochip 10. In some embodiments, the cavity, as viewed from above, may be square, rectangular, circular, round, polygonal, or another suitable shape. At least one opening 30 is defined on a bottom surface of the biochip 10 opposite the top surface and extending upward to a bottom of the cavity 20 such that at least a portion of the cavity 20 and the opening 30 bore vertically through the biochip 10. In some embodiments, a size of the cavity 20 size may be approximately 10 mm long, 6 mm wide, and 5 mm deep. As described herein, a longitudinal direction of the biochip 10 may be parallel to the length of the cavity 20 and a lateral direction of the biochip 10 may be parallel to the width of the cavity 20. The biochip 10 includes an inlet 40 and an outlet 50 each adapted for respectively receiving or discharging fluid, such as cell-culture media, and for transporting the same to or from the cavity 20. The terms “cell-culture media” and “culture media” may be used interchangeably herein. In some embodiments, the inlet 40 and outlet 50 are disposed on a same longitudinal end of the biochip 10.

[0022] Near the bottom of the cavity 20, adjacent to the opening 30, is a plurality of N > 2 of capillary tubes 100 arranged longitudinally cross the cavity 20 (N is an integer). The capillary tubes 100 are disposed within the cavity 20 and they extend from one side of the cavity 20 to an opposite side thereof, i.e., the capillary tubes 100 cross through the cavity 20. In some embodiments, the capillary tubes 100 are oriented parallel to each other. In some embodiments, there are two layers (e.g., rows) of capillary tubes 100, and each layer has 6 individual capillary tubes 100, for a total of 12 capillary tubes 100. In some embodiments, each capillary tube 100 has a square cross-section with outer width of 600 pm and an inner width of 500 pm; the spacing between adjacent outer surfaces of capillary tubes 100 is 400 pm; and an equivalent transverse cross-sectional density of capillary tubes is 1 capillary / mm2. Other cross-sectional shapes,arrangements, orientations, dimensions, and densities of the capillary tubes 100 may be utilized in some embodiments. For example: (i) some or all of the capillary tubes 100 may be arranged to extend longitudinally, laterally, and / or diagonally across the cavity 20; (ii) some or all of the capillary tubes 100 may be oriented parallel and / or at various angles to each other; (iii) some or all of the capillary tubes 100 may have varying cross-section shapes, inner sizes (e.g., widths or diameters), outer sizes (e.g., widths or diameters), and wall thicknesses; (iv) some or all of the capillary tubes 100 may have varying spacings therebetween; and (v) the transverse cross-sectional density of capillary tubes 100 may be approximately 0.5 to 10 capillaries / mm2. Arranging capillary tubes 100 into columns may be beneficial for observing cells and / or tissues therebetween, as viewed from below through the opening 30 via microscopy as indicated by FIG. 4, because lower capillary tubes 100 (closer to the opening 30) may not occlude the horizontal spaces between higher capillary tubes 100 (further away from the opening 30).

[0023] One or more perforated support ribs 110 may be disposed within the cavity 20 for supporting the capillary tubes 100 extending therethrough. The quantity of support ribs 110 may depend on, for example, the cross-sectional shapes and / or sizes, wall thicknesses, and materials of the capillary tubes 100 and / or a length of the cavity 20. For example, in an embodiment where cross-sectionally square capillary tubes 100 extend longitudinally across a 10 mm long cavity20 and each capillary tube 100 has an outer width of 100 pm, a wall thickness of 50 pm, and is constructed from polyethylene glycol, one support rib 110 may provide adequately support for the capillary tubes 100. In some embodiments consisting of a low quantity of capillary tubes 100 (e.g., two capillary tubes 100) or a low density of capillary tubes 100 (e.g., where the spacings between adjacent capillary tubes 100 is approximately an order of magnitude greater than the outer width of the capillary tubes 100), individual vertical and / or horizontal support members may be utilized to structurally support the capillary tubes in lieu of perforated support ribs 110.

[0024] In some embodiments, the biochip 10 is formed or printed from a biocompatible material that is ISO 10993 class II or above, such as polyethylene glycol (PEG, molecular weight 575 g / mol). Some materials may require a surface treatment to promote cell adhesion, which may be important for cell-culture experiments that need model cells to attach to and proliferate on a surface of the biochip 10. A poly-L-lysine solution may be used to coat one or more surfaces of the biochip 10, for example, one or more surfaces of the cavity 20.

[0025] FIGS. 2A-2B show an example of a flow of culture media in the biochip 10, where FIG. 2A is a cross section of the biochip 10 of FIG. 1A taken along line A-A and FIG. 2B is a cross section of the biochip 10 of FIG. 1A taken along line B-B. As shown in FIG. 2A, capillary tubes 100 are fluidically coupled in series from the inlet 40 to the outlet 50, with the capillary tubes 100 being connected by elbows 120 within body portions of the biochip 10 adjacent to the cavity 20 that redirect the flow of culture media. As shown in the FIG. 2A, the elbows 120 are U-shaped and non-capillary (e.g., walls of the elbows 120 are not formed or adapted for diffusion of solutes of a culture media). In some embodiments, some or all of the elbows 120 may be disposed within the cavity 20, in which case such elbows 120 may either be non-capillary or capillary-like (e.g., formed or adapted for diffusion of solutes of a culture media). In some embodiments, elbows 120 may be disposed outside the body portions of the biochip 10. In some embodiments, elbows 120 may follow a path that is other than U-shaped for redirecting a flow of culture media by approximately 180 degrees (e.g., exactly 180 degrees if a straight ingress capillary tube 100 to the elbow 120 and a straight egress capillary tube 100 from the elbow 120 are exactly parallel). For example, Q-shaped elbows 120 may redirect a flow of culture media approximately 360 degrees, such that the flow of culture media in an ingress capillary tube 100 to the elbow 120 and the flow of culture media in an egress capillary tube 100 from the elbow 120 is in a same direction. As another example, P-shaped elbows 120 may redirect a flow of culture media approximately 270 degrees, such that a flow of culture media in an ingress capillary tube 100 to the elbow 120 and the flow of culture media in an egress capillary tube 100 from the elbow 120 is are perpendicular (e.g., such capillary tubes 100 in the cavity 20 are perpendicular to each other and are disposed on different layers of the biochip 10).

[0026] In the embodiment shown in FIG. 2A, there are N=6 capillary tubes 100 in the layer of the biochip 10 that is shown by the cross section, and there are N-l=5 elbows 120 connecting each respective capillary tube 100 with its succeeding capillary tube 100. In general, given N capillary tubes 100, there are N-l elbows 120, where the ithelbow 120 connects the ithcapillary tube 100 to the (i+ 1 )thcapillary tube 100, where 1 < i < N and N is an integer.

[0027] In the embodiment shown in FIG. 2A, the capillary tubes 100 are arranged in a swirl pattern, such that culture media flows in a clockwise direction, from the inlet 40 to the outlet 50, flowing first within an outer track of the swirl pattern and flowing last within an inner track of the swirl pattern. As culture media flows within the capillary tubes 100, nutrients of the culturemedia diffuse out of the capillary tubes 100 and into a fluid within the cavity 20, for example, a culture-media bath therein. Thus, a concentration of nutrients in the culture media within outer tracks of the outside-in swirl pattern tends to be greater than a concentration of nutrients in the culture media within inner tracks of the outside-in swirl pattern. Consequently, an outside-in swirl pattern promotes uniform concentration of nutrients because more nutrient diffusion occurs through the outermost track, which neighbors only one other track (within a plane, or layer, of the swirl pattern, as described later) and an edge of the cavity 20, and less nutrient diffusion occurs through the innermost track, which neighbors two other tracks (within the plane, or layer, of the swirl pattern). In some embodiments, a swirl pattern may be counterclockwise.

[0028] FIG. 2B shows capillary tubes 100 arranged in two parallel layers of swirl patterns, where both layers direct a flow of culture in a clockwise direction. Although the layers may be physically parallel to each other, the term parallel as it is used in the preceding sentence refers to the concept of series versus parallel flows of culture media. In some embodiments, both layers of capillary tubes 100 are identical, and there is a 400 pm gap separating the layers. In some embodiments, there may be greater or fewer layers of capillary tubes 100, and each gap separating adj acent layers may be identical or they may be independent of other gaps separating other adj acent layers. Further, a direction of flow of culture media within each layer may be independent of any other layer (e.g., a first outside-in swirl pattern layer may direct culture media to flow in a clockwise direction and a second outside-in swirl pattern layer may direct culture media to flow in a counterclockwise direction). Still further, a flow pattern of each layer may be independent of a flow pattern of another layer, for example, a first layer may comprise capillary tubes 100 arranged in an outside-in swirl pattern and a second layer may comprise capillary tubes 100 arranged in an inside-out swirl pattern. Still further, other flow patterns of series-coupled capillary tubes 100 may be utilized besides swirl patterns, for example, capillary tubes 100 may be arranged in an S pattern (which may also be referred to as a zig-zag pattern or a sinusoidal pattern), where the flow of culture media within adjacent capillary tubes 100 of a given layer is in opposite directions. The biochip 10 includes appropriate channels for fluidically coupling the appropriate capillary tubes 100 to the inlet 40 and the outlet 50. In some embodiments, the elbows 120 may shaped, such that the flow of culture media through every capillary tube 100 in the cavity 20 (of the given layer) is in the same direction through the cavity 20. Q-shaped elbows 120 that extend from one end ofthe biochip 10 back to the other end of the biochip 10, effectively bypassing the cavity 20 on the “return” path of a flow of culture media.

[0029] The series coupling of capillary tubes 100 can be advantageous compared to parallel coupling. Parallel coupling may be achieved via a manifold disposed between an inlet and the capillary tubes that distributes culture media to all of the capillary tubes in parallel. Due to the small size of capillary tubes, manufacturing geometric tolerances can cause nontrivial differences in the cross-sectional areas, e.g., lumens, of the capillary tubes. Capillary tubes with smaller lumens restrict a flow of culture media compared to capillary tubes with larger lumens. Thus, in a parallel coupling of capillary tubes, manufacturing geometric tolerances can cause unequal flow of culture media and therefore unequal diffusion of nutrients within the cavity. In a series coupling of capillary tubes 100, the flow of culture media is, by definition, the same for the entire path from inlet 40 to outlet 50 (for a given layer of capillary tubes 100).

[0030] FIG. 3A shows a cross section of the biochip 10 of FIG. 1A taken along line C-C and FIG. 3B shows a cross section of the biochip 10 of FIG. 1A taken along line D-D. FIG. 3A shows a channel 130 that is fluidically coupled from an innermost capillary tube 100 of each layer of capillary tubes 100 to the outlet 50. FIG. 3B shows the support rib 110 supporting the two layers of capillary tubes 100. The support rib 110 is perforated to allow each capillary tube 100 to traverse therethrough. In some embodiments, each perforation has a diameter of 100 pm. In some embodiments of using the biochip 10 for building 3D cell-culture models, the support rib 110 may serve as a divider of the cavity 20 (and the fluid therein), such that cells on either side of the support rib 110 may be fluidically separated, and therefore, cells on one side of the support rib 110 may be of a different type than cells on another side of the support rib 110.

[0031] Diffusion of solutes across a membrane, such as a wall of a capillary tube 100, is proportional to a concentration gradient of solutes across the membrane. Thus, the wall thicknesses and / or materials of the capillary tubes 100 may be selected to allow for an appropriate rate of diffusion of certain solutes into and out of the capillary tubes 100, including metabolites and metabolic waste. For example, if the concentration of metabolic waste in the cavity 20 becomes too high (e.g., approximately 100 pmol / L), a healthy functioning of the cells in the cavity 20 may suffer. Metabolic waste may include toxins such as nitrogen compounds. Similarly, if the concentration of metabolites in the cavity becomes too low, then cells may fail to proliferate and / or grow, or cells may starve and / or die. It is therefore important, when using the biochip 10 forbuilding 3D cell-culture models, to supply the cavity 20 with sufficient metabolites and to continually remove metabolic waste.

[0032] In some embodiments, diffusion through the walls of the capillary tubes 100 (for one or both of metabolites and metabolic waste) may be significantly increased, e.g., by approximately a factor of 10, by adding micropores to the walls of the capillary tubes 100. In some embodiments, micropores having diameters of approximately 5 - 15 pm can be added to one or more of the walls (or portions thereof) of each capillary tube 100, with spacing between pores of 50- 300 pm, to significantly increase the rate(s) of diffusion. For example, in some embodiments, 10 pm micropores are added to all four surfaces of square capillary tubes 100 with a spacing between pores of 200 pm. In some embodiments, the diameters and / or density (spacings) of micropores maybe varied across capillary tubes 100 within a given layer or across capillary tubes 100 between layers to establish desired local concentrations of nutrients within a culture-media math within the cavity 20.

[0033] As culture media is transported within the capillary tubes 100, metabolites may diffuse out through the walls thereof, where they may be consumed by cells within the cavity 20. Accordingly, cells that consume metabolites may produce and release metabolic waste, which may diffuse in through the walls of the capillary tubes 100, where they may be transported thereby. The flow directions of culture media in the capillary tubes 100 is indicated by the arrows shown in FIGS. 2A-2B; capillary tubes 100 in both layers are adapted to transport fresh culture media that is rich in nutrients from the inlet 40 and into the culture-media bath within the cavity 20 (e.g., via diffusion), and the capillary tubes 100 are adapted to transport depleted (e.g., not fresh) culture media that is poor in nutrients and / or rich in metabolic waste to the outlet 50. In some embodiments, the flow rate within the capillary tubes 100 is on the order of several millimeters per second.

[0034] FIG. 4 shows a cross section of the 3D biochip of FIG. 1 A taken along line E-E as part of an example of a system 200 for studying cell biology and / or tissue engineering. The biochip 10 is disposed within a basin 210 having a transparent window 220 on a bottom surface thereof. The window 220 can be made of glass or clear plastic with a suitable thickness, for example, of 0.5 - 1 mm. At least a portion of the window 220 should overlap with a portion of the opening 30 such that it may be possible to visually observe a portion of the cavity 20 from beneath the system 200. A microscope 230 may be utilized for such observation. The basin 210 may contain a culture-media bath 280, such that the capillary tubes 100 may be immersed or submerged therein. Cells 290 are depicted by solid black ovals.

[0035] The inlet 40 (not visible in FIG. 4) may be coupled to an input port 240 of the basin 210 that is in fluidic communication with an inlet pump 250 for supplying fresh culture media. The inlet pump 250 may draw the fresh culture media from a fresh-storage reservoir (not illustrated). An inlet seal 260, for example an O-ring, may be disposed between the inlet 40 and the input port 240 to prevent leakage. The outlet 50 (not visible in FIG. 4) may be in fluidic communication with the cavity 20 of the biochip 10 to aid in circulation of the culture media. The outlet 50 may be coupled to an output port (not illustrated) of the basin 210 that is in fluidic communication with a reservoir.

[0036] A suction outlet 270 may be inserted into the cavity 20 from above, such that its lower tip is near the topmost layer of capillary tubes 100, for example, 50 pm - 3 mm above the topmost layer. The suction outlet 270, via negative pressure, can draw excessive used culture media of the culture-media bath 280 to a reservoir (not illustrated). The distance between the tip of the suction outlet 270 and the topmost layer of capillary tubes 100 can be controlled to build specific tissue-culture models. For example, in tissue-culture models that require an air-liquid interface, for example a skin-tissue model or a lung-tissue model, this distance can be 50 - 300 pm to enable the outer surface of the tissue model to be as close as possible to the air-liquid interface. As another example, for cancer-tissue models, the tissue-culture model can be submerged more deeply in the culture-media bath 280, allowing a distance between the tip of the suction outlet 270 and the topmost layer of capillary tubes 100 to be 500 pm - 3 mm.

[0037] In some embodiments, the outlet 50 may be fluidically coupled to a reservoir. In some embodiments, the outlet 50 may be fluidically coupled to the same reservoir to which the suction outlet 270 is fluidically coupled. In some embodiments, one, both, or the shared reservoir may be fluidically coupled to a waste treatment device (not illustrated) for separating metabolic waste from metabolites, and there may be a means for transporting the recovered metabolites to the inlet 40 for recirculation. For example, the waste treatment device may transport the recovered metabolites to a recovered-storage reservoir (not illustrated) from which recovered metabolites may be drawn via pumping or by gravity. In the case of pumping, the inlet pump 250 may transport the recovered and reprocessed metabolites from the recovered-storage reservoir to the inlet 40, oralternatively, a second pump may transport the recovered and reprocessed metabolites to the inlet 40 or to a fresh-storage reservoir.

[0038] The biochip 10 may be seeded with cells by a suitable manner. For example, seed cells may be suspended in a culture media and pipetted into the cavity 20 manually or in an automated and / or mechanized manner. Unfortunately, gravity may cause the initial seed cells to collect at the bottom of the cavity 20, adjacent to the window 220, instead of attaching to the capillary tubes 100. However, as more seed cells are pipetted into the cavity 20, at least some seed cells are expected to attach to the capillary tubes 100. Alternatively, seed cells may be suspended in a hydrogel pre-solution capable of gelation or polymerization, for example, upon exposure to heat or ultraviolet light. Gelation or polymerization causes the suspended seed cells to become encapsulated by the hydrogel, which prevents them from sinking to the bottom of the cavity 20 and collecting on the window 220. The hydrogel pre-solution may be exposed to heat or ultraviolet light after exiting the pipette and before entering the culture-media bath 280 within the cavity 20.

[0039] Once the biochip 10 is seeded with cells, it may be placed into an incubator (not illustrated) where environmental conditions may be controlled to promote (or to discourage) cell growth and / or proliferation. Controlled environmental conditions may include temperature, humidity, and CO2 concentration. Fresh, or input, culture media may be pumped into the inlet 40 and depleted, or output, culture media may be withdrawn from the outlet 50.

[0040] To further describe some embodiments in greater detail, reference is next made to an example of a technique 300 which may be performed by or using the biochip 10. FIG. 5 is a flowchart of an example of a technique for building a 3D cell-culture model. For simplicity of explanation, the technique 300 is depicted and described herein as a series of steps or operations. However, the steps or operations of the technique 300 in accordance with this disclosure can occur in various orders and / or concurrently. Additionally, other steps or operations not presented and described herein may be used. Furthermore, not all illustrated steps or operations may be required to implement a technique in accordance with the disclosed subject matter.

[0041] Referring to FIG. 5, the step 302 comprises immersing a 3D biochip into a culturemedia bath contained within a basin, the biochip comprising: a cavity on an upper surface of the biochip; N = 2 first capillary tubes fluidically coupled in series and crossing through the cavity, comprising a first initial capillary tube fluidically coupled to an inlet of the biochip and a first final capillary tube fluidically coupled to an outlet of the biochip; and N-l non-capillary first elbows,wherein each Ithelbow of the first elbows connects an ithcapillary tube of the first capillary tubes to an (i+l)thcapillary tube of the first capillary tubes, 1 = i < N.

[0042] The step 304 comprises seeding the cavity with cells.

[0043] The step 306 comprises incubating the cells.

[0044] The step 308 comprises pumping an input culture media into the inlet. In some embodiments, the technique 300 further comprises: inserting a suction outlet into the cavity such that its lower tip is near adjacent to at least one of the first capillary tubes; and applying a negative pressure to the suction outlet to draw culture media from the culture-media bath. In some embodiments, the technique 300 further comprises: capturing an output culture media discharged from the outlet; processing at least some of the output culture media with a waste treatment device to obtain a treated output culture media; and returning at least some of the treated output culture media to the inlet.

[0045] Some embodiments of the disclosed perfusion 3D cell culture include a biochip, comprising: a cavity on an upper surface of the biochip; N > 2 first capillary tubes fluidically coupled in series and crossing through the cavity, comprising a first initial capillary tube fluidically coupled to an inlet of the biochip and a first final capillary tube fluidically coupled to an outlet of the biochip; and N-l non-capillary first elbows, wherein each ilhelbow of the first elbows connects an ithcapillary tube of the first capillary tubes to an (i+ 1 )thcapillary tube of the first capillary tubes, 1 < i < N.

[0046] In some embodiments, the biochip further comprises: M > 2 second capillary tubes fluidically coupled in series and crossing through the cavity, comprising a second initial capillary tube fluidically coupled to the inlet of the biochip and a second final capillary tube fluidically coupled to the outlet of the biochip; and M-l non-capillary second elbows, wherein each kthelbow of the second elbows connects a kthcapillary tube of the second capillary tubes to a (k+ 1 )thcapillary tube of the second capillary tubes, 1 < k < M.

[0047] In some embodiments, the first capillary tubes are arranged in an outside-in swirl pattern.

[0048] In some embodiments, the first capillary tubes are arranged in one of the following: an outside-in swirl pattern; an inside-out swirl pattern; or an S pattern.

[0049] In some embodiments, the second capillary tubes are arranged in one of the following: an outside-in swirl pattern; an inside-out swirl pattern; or an S pattern.

[0050] In some embodiments, the first capillary tubes are arranged in different pattern than the second capillary tubes.

[0051] In some embodiments, the biochip further comprises: an opening defined on a bottom surface of the biochip adjoining the cavity.

[0052] In some embodiments, the biochip further comprises: at least one substantially vertical support rib that structurally supports at least one of the first capillary tubes.

[0053] In some embodiments, at least one of the first capillary tubes includes micropores.

[0054] In some embodiments, at least a portion of the biochip is 3D printed using projection micro stereolithography and two-photon polymerization.

[0055] Some embodiments of the disclosed perfusion 3D cell culture include a method, comprising: immersing a 3D biochip into a culture-media bath contained within a basin, the biochip comprising: a cavity on an upper surface of the biochip; N > 2 first capillary tubes fluidically coupled in series and crossing through the cavity, comprising a first initial capillary tube fluidically coupled to an inlet of the biochip and a first final capillary tube fluidically coupled to an outlet of the biochip; and N-l non-capillary first elbows, wherein each ithelbow of the first elbows connects an ithcapillary tube of the first capillary tubes to an (i+l)thcapillary tube of the first capillary tubes, 1 < i < N; seeding the cavity with cells; incubating the cells; and pumping an input culture media into the inlet.

[0056] In some embodiments of the method, the biochip further comprises: M > 2 second capillary tubes fluidically coupled in series and crossing through the cavity, comprising a second initial capillary tube fluidically coupled to the inlet of the biochip and a second final capillary tube fluidically coupled to the outlet of the biochip; and M-l non-capillary second elbows, wherein each kthelbow of the second elbows connects a kthcapillary tube of the second capillary tubes to a (k+ 1 )thcapillary tube of the second capillary tubes, 1 < k < M.

[0057] In some embodiments of the method, the first capillary tubes are arranged in one of the following: an outside-in swirl pattern; an inside-out swirl pattern; or an S pattern.

[0058] In some embodiments of the method, the second capillary tubes are arranged in one of the following: an outside-in swirl pattern; an inside-out swirl pattern; or an S pattern.

[0059] In some embodiments of the method, the method further comprises: inserting a suction outlet into the cavity such that its lower tip is near adjacent to at least one of the firstcapillary tubes; and applying a negative pressure to the suction outlet to draw culture media from the culture-media bath.

[0060] In some embodiments of the method, the method further comprises: capturing an output culture media discharged from the outlet; processing at least some of the output culture media with a waste treatment device to obtain a treated output culture media; and returning at least some of the treated output culture media to the inlet.

[0061] In some embodiments of the method, the biochip further comprises an opening defined on a bottom surface of the biochip adjoining the cavity; and the basin further comprises a transparent window on a bottom surface thereof.

[0062] In some embodiments of the method, the biochip further comprises at least one substantially vertical support rib that structurally supports at least one of the first capillary tubes.

[0063] In some embodiments of the method, at least one of the first capillary tubes includes micropores.

[0064] In some embodiments of the method, the basin further comprises: an input port fluidically coupled to the inlet; and an output port fluidically coupled to the outlet.

[0065] While several embodiments of the disclosure have been shown in the drawings, it is not intended that the disclosure be limited thereto, as it is intended that the disclosure be as broad in scope as the art will allow and that the specification be read likewise. Therefore, the above description should not be construed as limiting, but merely as exemplifications of particular embodiments.

Claims

WHAT IS CLAIMED IS:

1. A 3D biochip, comprising: a cavity on an upper surface of the biochip;N > 2 first capillary tubes fluidically coupled in series and crossing through the cavity, comprising a first initial capillary tube fluidically coupled to an inlet of the biochip and a first final capillary tube fluidically coupled to an outlet of the biochip; andN-l first elbows, wherein each ithelbow of the first elbows connects an ithcapillary tube of the first capillary tubes to an (i+l)thcapillary tube of the first capillary tubes, wherein 1 < i < N and N is an integer.

2. The biochip of claim 1, further comprising:M > 2 second capillary tubes fluidically coupled in series and crossing through the cavity, comprising a second initial capillary tube fluidically coupled to the inlet of the biochip and a second final capillary tube fluidically coupled to the outlet of the biochip; andM-l second elbows, wherein each kthelbow of the second elbows connects a kthcapillary tube of the second capillary tubes to a (k+l)thcapillary tube of the second capillary tubes, wherein 1 < k < M and M is an integer.

3. The biochip of claim 1, wherein: the first capillary tubes are arranged in an outside-in swirl pattern.

4. The biochip of claim 1, wherein the first capillary tubes are arranged in one of the following: an outside-in swirl pattern; an inside-out swirl pattern; or an S pattern.

5. The biochip of claim 2, wherein the second capillary tubes are arranged in one of the following: an outside-in swirl pattern; an inside-out swirl pattern; or an S pattern.

6. The biochip of claim 2, wherein: the first capillary tubes are arranged in different pattern than the second capillary tubes.

7. The biochip of claim 1, further comprising an opening defined on a bottom surface of the biochip adjoining the cavity.

8. The biochip of claim 1, further comprising at least one substantially vertical support rib that structurally supports at least one of the first capillary tubes.

9. The biochip of claim 1, wherein at least one of the first capillary tubes includes micropores.

10. The biochip of claim 1, wherein at least a portion of the biochip is 3D printed using projection micro stereolithography and two-photon polymerization.

11. A method of culturing cells, comprising: immersing a 3D biochip into a culture-media bath contained within a basin, the biochip comprising: a cavity on an upper surface of the biochip;N > 2 first capillary tubes fluidically coupled in series and crossing through the cavity, comprising a first initial capillary tube fluidically coupled to an inlet of the biochip and a first final capillary tube fluidically coupled to an outlet of the biochip; andN-l first elbows, wherein each ithelbow of the first elbows connects an ithcapillary tube of the first capillary tubes to an (i+ 1 )thcapillary tube of the first capillary tubes, wherein 1 < i < N and N is an integer; seeding the cavity with cells; incubating the cells; and pumping an input culture media into the inlet.

12. The method of claim 11, wherein the biochip further comprises:M > 2 second capillary tubes fluidically coupled in series and crossing through the cavity, comprising a second initial capillary tube fluidically coupled to the inlet of the biochip and a second final capillary tube fluidically coupled to the outlet of the biochip; andM-l second elbows, wherein each kthelbow of the second elbows connects a kthcapillary tube of the second capillary tubes to a (k+l)thcapillary tube of the second capillary tubes, wherein 1 < k < M and M is an integer.

13. The method of claim 11, wherein the first capillary tubes are arranged in one of the following: an outside-in swirl pattern; an inside-out swirl pattern; or an S pattern.

14. The method of claim 12, wherein the second capillary tubes are arranged in one of the following: an outside-in swirl pattern; an inside-out swirl pattern; or an S pattern.

15. The method of claim 11, further comprising: inserting a suction outlet into the cavity such that its lower tip is near adjacent to at least one of the first capillary tubes; and applying a negative pressure to the suction outlet to draw culture media from the culture-media bath.

16. The method of claim 11, further comprising: capturing an output culture media discharged from the outlet; processing at least some of the output culture media with a waste treatment device to obtain a treated output culture media; and returning at least some of the treated output culture media to the inlet.

17. The method of claim 11, wherein:the biochip further comprises an opening defined on a bottom surface of the biochip adjoining the cavity; and the basin further comprises a transparent window on a bottom surface thereof.

18. The method of claim 11, wherein the biochip further comprises at least one substantially vertical support rib that structurally supports at least one of the first capillary tubes.

19. The method of claim 11, wherein at least one of the first capillary tubes includes micropores.

20. The method of claim 11, wherein the basin further comprises: an input port fluidically coupled to the inlet; and an output port fluidically coupled to the outlet.

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